Eicosapentaenoic acid (referred to as “EPA” hereinafter) and docosahexaenoic acid (referred to as “DHA” hereinafter), which are categorized as n-3 polyunsaturated fatty acids, have a variety of biological effects, and are used as medical products, health food products, food product materials, and the like. EPA ethyl ester is used as a therapeutic agent for treating arteriosclerosis and hyperlipidemia. Further, beverages to which fish oil containing EPA and DHA has been added have been approved as a food for specified health uses. Furthermore, demand for these fatty acids as a supplement is very high in Japan and other countries.
Since PUFAs have many double bonds, they are very unstable to oxidation. Therefore, in a process for preparing PUFA-containing oil and fat, it is very desirable to use an enzyme reaction that proceeds under mild conditions, such as at room temperature and normal pressure.
It has been known that some of lipase products for industrial use, which are obtained mainly from microorganisms, have a property of less reacting with PUFAs. PUFA-concentrated oil and fat can be produced by dominantly liberating shorter-chain fatty acids using lipase having such a property, followed by removal of the free fatty acids. For instance, a process for preparing DHA-concentrated oil and fat by hydrolysis of tuna oil using a lipase derived from Candida cylindracea followed by removal of the free fatty acids has been reported (Patent Document 1).
It has been known that for enzyme reactions even in an organic solvent, water plays an important role on enzyme activation (non-Patent Document 1). It has been reported that when PUFAs are concentrated from cod liver oil by alcoholysis, which is a reaction to eliminate fatty acids from glycerides by treating with alcohol, and addition of water promotes the lipase reaction (Non-Patent Document 2). On the other hand, it has been reported that such an alcoholysis reaction of oil and fat proceeds under virtually anhydrous conditions with certain lipases (Patent Document 2). Nevertheless, the amount of lipase used was very high at 10% relative to the amount of oil, and the lipase must be immobilized to improve productivity.
Alcoholysis reaction carried out with lower alcohols yields fatty acid lower alcohol ester, which can be easily removed by distillation or the like.    [Patent Document 1] JP 58-165796 A    [Patent Document 2] JP 9-510091 A    [Non-Patent Document 1] J. S. Dordick, “Enzymatic catalysis in monophasic organic solvents”, Enzyme Microb. Technol., 1989, 11, April, 194-211.    [Non-Patent Document 2] L. Zui and O. P Ward, “Lipase-catalyzed alcoholysis to concentrate the n-3 polyunsaturated fatty acids of cod liver oil”, Enzyme Microb. Technol., 1993, 15, Jul., 601-606.